Structural Biology of Multi-Domain Proteins and Multi-Protein Machinery in DNA Replication and Repair

DNA 复制和修复中多域蛋白和多蛋白机制的结构生物学

• 批准号: 10382072
• 负责人: WALTER J. CHAZIN
• 金额: $ 3.7万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10382072
• 关键词: Address; Amino Acid Sequence; Architecture; Bacteria; Biochemical; Biological; Biological Models; Cells; Complement; Complex; DNA; DNA Damage; DNA Primase; DNA Primers; DNA Repair; DNA Replication Damage; DNA biosynthesis; DNA replication fork; DNA-Directed DNA Polymerase; Defect; Development; Disease; Genome; Genomic Instability; Goals; Hand; Human; Knowledge; Lead; Length; Lower Organism; Maintenance; Malignant Neoplasms; Modernization; Multiprotein Complexes; Mutation; Patients; Phase; Polymerase; Process; Proteins; RNA; Replication Origin; Research; Role; SS DNA BP; Signaling Protein; Simian virus 40; Single-Stranded DNA; Structural Protein; Structure; Tertiary Protein Structure; Validation; base; chemotherapy; helicase; human DNA; mutant; protein structure; recruit; replication factor A; response; structural biology; targeted treatment; three dimensional structure; tool

PROJECT SUMMARY Faithful replication of DNA and response to damaged DNA, essential to cell propagation and survival, proceeds via the action of multi-protein machines. While considerable progress has been made in elucidating the mechanisms of DNA replication and damage response from studies of bacteria and other lower organisms, information on humans is lacking because the protein sequences and structures are not conserved. Our long- term goals are to understand the action of human DNA replication and damage response machinery. Our strategy is to elucidate the structural basis for these processes using the full complement of modern structural biology tools and interpret this information using a range of biochemical and biological approaches. We have shown in the SV40 model system that the helicase that creates the single-stranded DNA (ssDNA) at the origin of replication also actively loads the human ssDNA binding protein replication protein A (RPA), and that this action is essential for DNA priming by DNA polymerase -primase (pol-prim). We have also characterized the complex and dynamic structural architectural of the modular RPA protein as it engages this ssDNA template and have begun to address the quandary of how RPA releases the template to hand off to pol-prim. Once the primase subunits are loaded on the DNA template, an ~10 nt RNA is synthesized and the primed template is transferred to the pol  subunits for extension to the final ~30 nt of RNA-DNA primer. Our goal is to continue to define the trajectory of actions on the template as it is replicated. The next phase of our research is to solve the fundamental mysteries about the release of RPA and loading of pol-prim onto the template, the counting of primer length by primase, and the hand-offs from primase to pol  and in turn to pol  or pol . Our DNA damage response research seeks to understand the mechanism of action of the key proteins involved, discern the biochemical bases for malfunctions caused by disease-associated mutations, and ultimately use this knowledge to help evaluate the potential of chemotherapies targeted to these proteins. Here again, RPA has a critical role, in this case, in recruiting partner proteins that stall and remodel replication forks and activate important signaling proteins. The goal is to obtain structural understanding and functional validation of the interactions between RPA and damage response proteins, and their role in responding to encounters with damaged DNA.

项目概要 DNA 的忠实复制和对受损 DNA 的反应，对于细胞繁殖和生存至关重要 通过多蛋白质机器的作用，在阐明这一点方面已经取得了相当大的进展。 来自细菌和其他低等生物研究的 DNA 复制和损伤反应机制， 由于蛋白质序列和结构不保守，因此缺乏有关人类的信息。 术语目标是了解人类 DNA 复制和损伤反应机制的作用。 策略是利用现代结构的完整补充来阐明这些过程的结构基础 我们拥有生物学工具并使用一系列生化和生物学方法来解释这些信息。 SV40 模型系统显示，在起始点产生单链 DNA (ssDNA) 的解旋酶 复制过程还主动加载人类 ssDNA 结合蛋白复制蛋白 A (RPA)，并且这 DNA 聚合酶 α-primase (pol-prim) 的作用对于 DNA 引发至关重要。 模块化 RPA 蛋白与 ssDNA 模板结合时的复杂且动态的结构体系 已经开始解决 RPA 如何发布模板以将其移交给 pol-prim 的难题。 将引物酶亚基加载到 DNA 模板上，合成约 10 nt RNA，并将引物模板 转移到 pol  亚基以延伸至 RNA-DNA 引物的最终约 30 nt。 定义复制模板上的动作轨迹。我们研究的下一阶段是解决这个问题。 关于 RPA 释放和 pol-prim 加载到模板上的基本奥秘，计数 引物长度，以及从引物酶到 pol  的交接，然后再到 pol  或 pol  我们的 DNA 损伤。 反应研究旨在了解所涉及的关键蛋白质的作用机制，辨别 疾病相关突变引起的功能障碍的生化基础，并最终利用这些知识 帮助评估针对这些蛋白质的化疗的潜力，RPA 再次发挥着关键作用， 在这种情况下，在招募伙伴蛋白时，它们会阻止和重塑复制叉并激活重要的信号传导 目标是获得蛋白质之间相互作用的结构理解和功能验证。 RPA 和损伤反应蛋白，以及它们在应对受损 DNA 时的作用。